title: Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA).
abstract: An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.
authors.affiliation: Division of Pathway Medicine, Medical School, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK. till.bachmann@ed.ac.uk.
authors.forename: D K
authors.initials: DK
authors.lastname: Corrigan
authors.forename: H
authors.initials: H
authors.lastname: Schulze
authors.forename: G
authors.initials: G
authors.lastname: Henihan
authors.forename: A
authors.initials: A
authors.lastname: Hardie
authors.forename: I
authors.initials: I
authors.lastname: Ciani
authors.forename: G
authors.initials: G
authors.lastname: Giraud
authors.forename: J G
authors.initials: JG
authors.lastname: Terry
authors.forename: A J
authors.initials: AJ
authors.lastname: Walton
authors.forename: R
authors.initials: R
authors.lastname: Pethig
authors.forename: P
authors.initials: P
authors.lastname: Ghazal
authors.forename: J
authors.initials: J
authors.lastname: Crain
authors.forename: C J
authors.initials: CJ
authors.lastname: Campbell
authors.forename: K E
authors.initials: KE
authors.lastname: Templeton
authors.forename: A R
authors.initials: AR
authors.lastname: Mount
authors.forename: T T
authors.initials: TT
authors.lastname: Bachmann
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D055664:Electrochemical Techniques; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus; D019095:Point-of-Care Systems / Q000592:standards*; D016133:Polymerase Chain Reaction; D013203:Staphylococcal Infections / Q000175:diagnosis*